Effect of temperature on lipase-catalyzed esterification in organic solvent

Summary For the esterification of 2-(4-ethylphenoxy)propionic acid catalyzed by lipase MY (Candida rugosa) in isopropyl ether containing a suitable amount of water, the enantioselectivity for the reaction has become higher as the reaction temperature increasing. In contrast, the reverse trend of the temperature effect has been observed for lipase AY (Candida rugosa). A model for these temperature dependence has been proposed.     

Lipases are soluble and active in glycerol carbonate as a novel biosolvent

Glycerol carbonate was synthesized as biosolvent for the development of soluble enzymatic system. The effects of various reaction parameters on activity and stability of lipases were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. Enzymatic activity in glycerol carbonate was compared with that in water and in conventional organic solvents with different ionizing and dissociating abilities. The pK_a value of trichloroacetic acid and transesterification activities of Candida antarctica lipase B and Candida rugosa lipase in glycerol carbonate are similar to those in water, indicating that ionizing and dissociating powers are capable of satisfactorily predicting the biocompatibility of organic solvents for soluble enzymatic systems.     

Immobilization of lipase within carbon nanotube–silica composites for non-aqueous reaction systems

The immobilization of lipases within sol–gel derived silica, using multi-walled carbon nanotubes (MWNTs) as additives in order to protect the inactivation of lipase during sol–gel process and to enhance the stability of lipase, was investigated. Three sol–gel immobilized lipases (Candida rugosa, Candida antarctica type B, Thermomyces lanuginosus) with 0.33% (w/w) MWNT showed much higher activities than lipase immobilized without MWNT. The influence of MWNT content and MWNT shortened by acid treatment in the sol–gel process on the activity and stability of immobilized C. rugosa lipase was also studied. In hydrolysis reaction, immobilized lipase containing 1.1% pristine MWNT showed 7 times higher activity than lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT showed 10 times higher activity in esterification reaction, compared with lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT retained 96% of initial activity after 5 times reuse, while the lipase immobilized without MWNT was fully inactivated under the same condition.     

Isomerase activity of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase in the optimized conversion of racemic ibuprofen to (<Emphasis Type="Italic">S</Emphasis>)-ibuprofen

The Candida rugosa lipase catalyzed Dynamic Kinetic Resolution of racemic ibuprofen methyl ester produced (S)-ibuprofen in over 90% yield within 72 h at pH 7.6. The best concentration of various buffers for these reactions ranged from 0.2 to 0.5 M. The commercial lipase was found to be acidic altering the final pH of the reaction mixtures. Dimethylformamide co-solvent maintained the reaction pH better than dimethylsulfoxide. Lower concentrations of ibuprofen methyl ester and higher stirring rates led to faster conversions. The minimal amount of lipase needed was 20 mg/mL buffer. Reaction of (R)-ibuprofen methyl ester under the optimized conditions excluding the lipase led to no racemization, indicating that the conversion of (R)-ibuprofen methyl ester to (S)-ibuprofen is catalyzed by the enzyme, thus, indicating Candida rugosa lipase possess Isomerase activity.     

High-yield synthesis of wax esters catalysed by modified <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

Candida rugosa lipase (CRL) was applied in a non-solvent esterification reaction to yield twelve wax esters. All products were obtained in nearly 100% yield for 10 h at 50°C when immobilized PEG₂₀₀₀-activated C. rugosa lipase was added to the reaction mixture. The surfactant had also a beneficial effect on the stability of the biocatalytic preparation with 83% of its activity conserved after the seventh run of repeated batch reactions.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

Efficient microbial elimination of methanol inhibition for Naproxen resolution by a lipase

To eliminate methanol inhibition of the activity of a lipase, methanotrophic bacteria, which can convert methanol into water and CO₂, were introduced to the reaction of enantioselective hydrolysis of Naproxen methyl ester catalysed by lipase from Candida rugosa. Both the activity and stability of lipase were improved by the removal of methanol by the bacteria.     

A magnetically separable biocatalyst for resolution of racemic naproxen methyl ester

Candida rugosa lipase (CRL) was encapsulated via the sol–gel method, using 5, 11, 17, 23-tetra-tert-butyl-25,27-bis(2-aminopyridine)carbonylmethoxy-26, 28-dihydroxy-calix[4]arene-grafted magnetic Fe₃O₄ nanoparticles (Calix-M-E). The catalytic activity of encapsulated lipase (Calix-M-E) was tested both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic naproxen methyl ester. The present study demonstrated that the calixarene-based compound has the potential to enhance both reaction rate and enantioselectivity of the lipase-catalyzed hydrolysis of racemic naproxen methyl ester. The encapsulated lipase (Calix-M-E) had great catalytic activity and enantioselectivity (E > 400), as well as remarkable reusability as compared to the encapsulated lipase without supports (E = 137) for S-Naproxen.     

A new method for improving the enantioselectivity of lipase-catalyzed hydrolysis in organic solvent containing a small amount of water in the presence of metal ions

The hydrolysis of racemic butyl 2-(4-substituted phenoxy)propionates having various substituents catalyzed by lipase MY from Candida rugosa was achieved in di-isopropyl ether containing 0.75% (v/v) of 2.4 M LiCl or 1.2 M MgCl₂ aqueous solution. Water molecules hydrated to the metal ion in isopropyl ether acted as a nucleophile to cause the hydrolysis of these esters as with water alone. Metal ions used significantly enhanced their enantioselectivities by 100-fold or above, as compared with the ordinary reaction media.     

Chemical modification of lipases with various hydrophobic groups improves their enantioselectivity in hydrolytic reactions

Semi-purified lipases from Candida rugosa, Pseudomonas cepacia and Alcaligenes sp. were chemically modified with a wide range of hydrophobic groups such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl, lauroyl and acetyl moieties. The Candida rugosa lipase MY modified with the benzyloxycarbonyl group (modification ratio = 84%) brought about a 15-fold increase in enantioselectivity (E value) towards the hydrolysis of racemic butyl 2-(4-ethylphenoxy)propionate in an aqueous buffer solution, although the enzymatic activity was decreased. The origin of the enantioselectivity enhancement by chemical modification of the lipase is attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer.     

Improved stability of the lipase from Candida rugosa in different purification degrees by chemical modification

Summary Semipurified lipase of Candida rugosa and pure isoforms (lipase A and lipase B) have been chemically modified using two methodologies based on polyethyleneglycol (PEG). The activation of PEG with p-NO₂-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG in the enzymatic activity of the lipase. The chemical modification increases the stability of pure lipases in isooctane at 50 °C.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Studies of reaction parameters on synthesis of Citronellyl laurate ester via immobilized Candida rugosa lipase in organic media

Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.     

Dependence of Lipase Activity on Water Content and Enzyme Concentration in Reverse Micelles

The activity of Candida rugosa lipase (EC 3.1.1.3) in reverse micelles has been measured at various concentrations of water and enzyme with the aim of answering the question, why is the enzyme activity affected by the molar ratio of water to surfactant (w₀ = [H₂O]/[Surfactant])? In the low range of water content (below w₀ ≈ 6), the activity increases with increasing water content, indicating the requirement of a minimum amount of water for the full expression of enzymatic activity. The minimal w₀-value for obtaining maximal activity depends on the enzyme concentration: The higher the enzyme concentration, the higher w_{0, max}. In addition, it was found that, at least for the case of Candida rugosa lipase, the measured dependence of enzyme activity on w₀ does not represent a true chemical equilibrium. Changing the w₀-value during the reaction does not change the activity as expected on the basis of the w₀-activity profile obtained for single w₀ point measurements. All these observations, however, cannot be directly generalized to all enzymes in reverse micelles, due to the peculiarity of lipase. In particular, the enzyme seems to inactivate irreversibly during the solubilization process.     

Increasing the catalytic efficiency of Candida rugosa lipase for the synthesis of tert-alkyl butyrates in low-water media

Abstract

Reactions involving tert-alcohols and their esters are generally not catalyzed by lipases. Candida rugosa lipase is one of the few lipases which shows at least limited catalytic activity towards tert-alcohols and their esters. Using transesterification of tributyrin with tertiary butyl and amyl alcohols as a model reaction, it is shown that precipitation of lipase by a tertiary alcohol in the presence of a buffer with optimum concentration enhances the catalytic activity 7 fold as compared to rates obtained with lyophilized powders. Optimization of the ratio of triglyceride to tert-alcohols and medium engineering gave an initial rate which was 41 times higher than that obtained with lyophilized powders. Hence, use of a simple enzyme formulation, coupled with optimization of reaction conditions led to Candida rugosa lipase becoming a useful catalyst for catalyzing transesterification involving tertiary alcohols.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Enhanced catalytic ability of Candida rugosa lipase immobilized on pore-enlarged hollow silica microspheres and cross-linked by modified dextran in both aqueous and non-aqueous phases

Candida rugosa lipase (CRL) is one of the most widely used lipases. To enhance the catalytic abilities of CRL in both aqueous and non-aqueous phases, hollow silica microspheres (HSMSs) with a pore size of 18.07 nm were used as an immobilization support, and aldehydecontaining dextrans were employed to further cross-link the adsorbed CRL. In the experimental ranges examined, the loading amount of lipase linearly increased to 171 ± 3.4 mg_protein/g_support with the CRL concentration and all the adsorption equilibriums were reached within 30 min. After simple cross-linking, the tolerance to pH 4.0 ～ 8.0 as well as the thermal stability of immobilized CRL at 40 ～ 80°C were both substantially increased, and 82 ± 2.1% activity remaining after the sixth reuse. The immobilized CRL was successfully applied to the resolution of racemic ibuprofen in non-aqueous phase. The initial reaction rate increased by 1.4- and 3.6-fold compared with the rates of adsorbed and native lipases, respectively. Furthermore, the R-ibuprofen was obtained at ee > 93%, and the enantiomeric ratio reached E > 140 at the conversion of 50 ± 1.5% within 48 h.     

Enantioselective hydrolysis of rasemic naproxen methyl ester with sol–gel encapsulated lipase in the presence of sporopollenin

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, the Candida rugosa lipase (CRL) was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) in the presence and absence of sporopollenin and activated sporopollenin as additive. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of rasemic Naproxen methyl ester that was studied in aqueous buffer solution/isooctane reaction system. The results indicated that the sporopollenin based encapsulated lipase particularly had higher conversion and enantioselectivity compared to the sol–gel free lipase. In this study, excellent enantioselectivity (E > 400) has been noticed for most lipase preparations (E = 166 for the free enzyme) with an ee value ～98% for S-Naproxen. Moreover, (S)-Naproxen was recovered from the reaction mixture with 98% optical purity.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.